Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum
Griffiths, Scott A. ; Cox, Russell J. ; Overdijk, Elysa J.R. ; Mesarich, Carl H. ; Saccomanno, Benedetta ; Lazarus, Colin M. ; Wit, Pierre J.G.M. de; Collemare, Jérôme - \ 2018
PLoS ONE 13 (2018)12. - ISSN 1932-6203

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.

Specific Hypersensitive Response–Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato
Mesarich, Carl H. ; Ӧkmen, Bilal ; Rovenich, Hanna ; Griffiths, Scott A. ; Wang, Changchun ; Karimi Jashni, Mansoor ; Mihajlovski, Aleksandar ; Collemare, Jérôme ; Hunziker, Lukas ; Deng, Cecilia H. ; Burgt, Ate Van Der; Beenen, Henriek G. ; Templeton, Matthew D. ; Bradshaw, Rosie E. ; Wit, Pierre J.G.M. De - \ 2018
Molecular Plant-Microbe Interactions 31 (2018)1. - ISSN 0894-0282 - p. 145 - 162.
Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta–induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X–based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.
Down-regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato : A secondary metabolite prevents fungal biotrophy
Griffiths, Scott ; Mesarich, Carl H. ; Overdijk, Elysa J.R. ; Saccomanno, Benedetta ; Wit, Pierre J.G.M. De; Collemare, Jérôme - \ 2018
Molecular Plant Pathology 19 (2018)2. - ISSN 1464-6722 - p. 369 - 380.
Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during colonisation of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but they are repressed during the biotrophic phase of tomato colonization. It was suggested that tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, while retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato similar to wild type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low temperature stress. Overall this study demonstrates that repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, while its production is important for survival outside its host.
Novel effectors identified in the apoplast of Cladosporium fulvum-infected tomato
Mesarich, C. ; Ökmen, B. ; Rövenich, H.J. ; Karimi Jashni, M. ; Wang, C. ; Griffiths, S.A. ; Collemare, J.A.R. ; Deng, C. ; Wit, P.J.G.M. de - \ 2016
Tomato leaf mold disease is caused by the biotrophic fungal pathogen Cladosporium fulvum. To colonize the leaf apoplast, C. fulvum secretes a collection of effector proteins that modulate host immune responses, as well as other proteins (e.g., carbohydrate-active enzymes or CAZys) that facilitate nutrient acquisition. In the presence of cognate Cf immune receptors, however, many of these proteins trigger immune responses that render the pathogen avirulent. Characterization of the C. fulvum apoplastic secretome is required to further understand the abovementioned processes, and to identify novel sources of resistance against this pathogen. We have used liquid chromatography–tandem mass spectrometry (LC–MS/MS) to identify 141 secreted and surface-associated fungal proteins present in apoplastic fluid harvested from compatible C. fulvum–tomato interactions. In addition to the known effectors identified in previous studies, this collection contains >70 new C. fulvum candidate effector (CfCE) proteins. Using a Potato virus X (PVX)-based expression system, we show that nine of these CfCEs, including Ecp11-1, which has homology to AvrLm3 and AvrLmJ1 of Leptosphaeria maculans, trigger cell death in particular wild accessions of tomato. Thus, our study has likely uncovered novel avirulence effectors of C. fulvum, as well as Cf immune receptors in wild tomato with new specificities against this pathogen. An overview of the C. fulvum apoplastic secretome will be presented.
Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization
Griffiths, Scott ; Mesarich, Carl H. ; Saccomanno, Benedetta ; Vaisberg, Abraham ; Wit, Pierre J.G.M. de; Cox, Russell ; Collemare, Jérôme - \ 2016
Proceedings of the National Academy of Sciences of the United States of America 113 (2016)25. - ISSN 0027-8424 - p. 6851 - 6856.
Cytoxicity - Emodin - Gene cluster - Nataloe-emodin - Secondary metabolism

Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum. A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families.

The secondary metabolome of the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit; Pedro Crous, co-promotor(en): Jerome Collemare. - Wageningen : Wageningen University - ISBN 9789462575813 - 167
passalora fulva - secundaire metabolieten - metabolomen - genen - genomica - biologische activiteit - biosynthese - natuurlijke producten - passalora fulva - secondary metabolites - metabolomes - genes - genomics - biological activity - biosynthesis - natural products

Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised
by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other
organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal
growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived
from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats.
Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous
fungi contain very large numbers of genes related to SM production. Yet these genes are typically
silent under laboratory conditions. There are now many tools and strategies available to activate
or clone silent SM genes. This thesis details our efforts to apply various methods to define and
then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato
containing many silent SM genes and gene clusters.

In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi
are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM
biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and
increase the chemical diversity of fungi are detailed. These include the deletion or over-expression
of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation
of growth conditions. Heterologous expression of silent SM genes in a production host is also
discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced
as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis
showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10
polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid
and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the
majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole
detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to
apply the aforementioned strategies to induce these silent genes and obtain new compounds. The
importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential
medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential
source of novel biosynthetic enzymes.

In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was
analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic
genes linked to SM production, such as those encoding decorating enzymes and regulators. Products
of these SM genes or gene clusters were speculated, based on their similarity to those characterized
in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other
fungal species. Remarkably, two predicted functional gene clusters were linked to the production
of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive
oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum
under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR
and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous
findings, the majority of SM genes remained silent and only cladofulvin was detected. During
growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were
significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We
confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into
their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our
studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent
another mechanism associated with a biotrophic lifestyle.

In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim
of discovering new compounds. Many Ascomycete-specific global regulators of SM production
and morphological development in other fungi were identified in C. fulvum. We investigated
three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes
encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis
was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of
cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild
type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was
affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly
repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of
VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was
not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin.
This suggests that cladofulvin production is not required for asexual reproduction. The main
finding of this chapter is that global regulator manipulation cannot considered to be a universal
tool to discover new fungal natural products.

In chapter 4, anthraquinones and closely related compounds such as anthrones, anthracyclines
and xanthones are considered. Emodin is perhaps the most well characterised anthraquinone that
is produced by many fungi and plants. Once synonymous only with constipation, this former
laxative has since been investigated for its useful anti-cancer, anti-diabetic, anti-infective and antiinflammatory properties. Cladofulvin is a homodimeric anthraquinone composed of nataloe-emodin joined in a remarkably asymmetrical configuration. Dimeric anthraquinones and xanthones are also bioactive, most commonly tested for anti-infective and anti-cancer activities. Despite the ubiquity and medicinal qualities of anthraquinones and related compounds, very few of their biosynthetic pathways are known. No enzymes capable of dimerizing anthraquinones had yet been identified. In this chapter we demonstrated that cladofulvin was very cytotoxic towards human cancer cell-lines, crucially, up-to 300-fold more than its monomeric precursor nataloe-emodin against certain celllines. This became an added incentive to elucidate the cladofulvin pathway and identify the enzyme responsible for dimerizing nataloe-emodin. We confirmed earlier predictions that PKS6/claG is the core gene which starts cladofulvin biosythesis. Deletion of claG abolished cladofulvin production
and no related metabolites were observed. A route to cladofulvin biosynthesis was proposed, guided
by the work performed on the monodictyphenone biosynthetic pathway in Aspergillus nidulans.

We predicted early acting cladofulvin genes and cloned them for heterologous expression in A.
oryzae strain M-2-3. Using this approach we were able to confirm the first five genes in cladofulvin
biosynthesis, claBCFGH, which yielded a reduced and dehydrated form of emodin. This is the
point at which the pathways to cladofulvin and monodictyphenone production diverge. It was
speculated that this emodin-related intermediate might be converted into nataloe-emodin by claK
and/or claN. Finally, it was confirmed that the final step in the cladofulvin pathway is encoded by
claM. Targeted deletion of claM yielded a mutant that accumulated nataloe-emodin and emodin
but no cladofulvin. We discuss how the sequence of claM and ClaM will accelerate the discovery
of functionally similar genes and enzymes, providing a template to engineer enzymes capable of
forming novel dimers from existing monomers.

In chapter 5 the natural role of cladofulvin was considered. This SM is consistently produced by
C. fulvum and global regulator mutants in vitro. The respective biosynthetic genes appear most
active during early and late stages of infection of tomato, but are down-regulated during biotrophic
growth phase (chapter 2). The Δclag mutants (chapter 3) were not overtly different from the wild
type during growth in vitro. We inoculated tomato plants with this mutant in order to test whether
or not cladofulvin was required for normal infection. Simultaneously, we inoculated a C. fulvum
transformant carrying an extra copy of the cladofulvin pathway-specific relulator, OE.claE, fused
to the promoter region of the Avr9 effector gene. The strain was expected to produce cladofulvin
once the fungal hyphae penetrate host stomata and begin to colonise the apoplastic space. In this
way, we aimed to test the effect of cladofulvin over-production on disease symptom development.
The growth of each strain on tomato plants was monitored by RT-qrtPCR at 4, 8 and 12 days post
inoculation (dpi). At each time point the infections were inspected microscopically to detect any
phenotypic abnormalities. We report that the loss of claG did not result an abnormal infection.
Both wild type and ΔclaG mutants sporulated without causing necrosis or dessication of host leaves.
In distinct contrast, brown spots appeared on leaves infected by the OE.claE transformant between
8 – 12 dpi. This was accompanied by much stronger fungal growth and significant accumulation
of cladofulvin. The leaves became desiccated and brittle before the fungus conidiated. Possible
reasons for this phenotype are discussed. A small suite of in vitro experiments was performed on the
Δclag and wild type strains in order to test the role of cladofulvin in survival. Consistent with the
absence of a photoprotective pigment, Δclag spores were considerably more sensitive to ultraviolet
(UV) radiation. Suggesting a role in protection against low temperatures, Δclag spores were less
resistant to repeated cycles of freezing and thawing. Cladofulvin biosynthesis was stimulated and
repressed by cold and heat shocking mature C. fulvum colonies, respectively. Altogether, these
results suggested that cladofulvin confers resistance to abiotic stress.

In chapter 6 the results obtained in this thesis are discussed in a broader context. Particularly,
the discovery of the cytochrome P450 that is involved in dimerization of anthraquinones might
enable discovery of homologous genes encoding enzymes with different specificities. Combining
bioinformatic and functional analyses should prove to be a powerful strategy for discovering
compounds with new biological activities, or enzymes relevant to metabolic engineering.

Regulation of secondary metabolite production in the fungal tomato pathogen Cladosporium fulvum
Griffiths, S.A. ; Saccomanno, B. ; Wit, P.J.G.M. de; Collemare, J. - \ 2015
Fungal Genetics and Biology 84 (2015). - ISSN 1087-1845 - p. 52 - 61.
Cladosporium fulvum is a non-obligate biotrophic fungal tomato pathogen for which fifteen secondary metabolite (SM) gene clusters were previously identified in its genome. However, most of these SM biosynthetic pathways remain cryptic during growth in planta and in different in vitro conditions. The sole SM produced in vitro is the pigment cladofulvin. In this study, we attempted to activate cryptic pathways in order to identify new compounds produced by C. fulvum. For this purpose, we manipulated orthologues of the global regulators VeA, LaeA and HdaA known to regulate SM biosynthesis in other fungal species. In C. fulvum, deleting or over-expressing these regulators yielded no new detectable SMs. Yet, quantification of cladofulvin revealed that CfHdaA is an activator whilst CfVeA and CfLaeA seemed to act as repressors of cladofulvin production. In the wild type strain, cladofulvin biosynthesis was affected by the carbon source, with highest production under carbon limitation and traces only in presence of saccharose. Repression of cladofulvin production by saccharose was dependent on both CfVeA and CfLaeA. Deletion of CfVeA or CfLaeA caused production of sterile mycelia, whilst ¿cfhdaa deletion mutants sporulated, suggesting that cladofulvin production is not linked to asexual reproduction. Profiling the transcription of these regulators showed that CfHdaA-mediated regulation of cladofulvin production is independent of both CfVeA and CfLaeA. Our data suggest CfLaeA directly affects cladofulvin production whilst the effect of CfVeA is indirect, suggesting a role for CfLaeA outside of the Velvet complex. In conclusion, our results showed that regulation of SM production in C. fulvum is different from other fungi and indicate that manipulation of global regulators is not a universal tool to discover new fungal natural products.
Novel mutations detected in avirulence genes overcoming tomato Cf resistance genes in isolates of a Japanese population of Cladosporium fulvum
Iida, Y. ; Hof, P.M.J. van 't; Beenen, H.G. ; Mesarich, C.H. ; Kubota, M. ; Stergiopoulos, I. ; Mehrabi, A. ; Notsu, A. ; Fujiwara, K. ; Bahkali, A. ; Abd-Elsalam, K. ; Collemare, J. ; Wit, P.J.G.M. de - \ 2015
PLoS ONE 10 (2015)4. - ISSN 1932-6203 - 18 p.
fungal effector proteins - leaf mold - disease resistance - virulence factor - cf-4-mediated resistance - allelic variation - passalora-fulva - plant-pathogen - binding-site - avr4
Leaf mold of tomato is caused by the biotrophic fungus Cladosporium fulvum which complies with the gene-for-gene system. The disease was first reported in Japan in the 1920s and has since been frequently observed. Initially only race 0 isolates were reported, but since the consecutive introduction of resistance genes Cf-2, Cf-4, Cf-5 and Cf-9 new races have evolved. Here we first determined the virulence spectrum of 133 C. fulvum isolates collected from 22 prefectures in Japan, and subsequently sequenced the avirulence (Avr) genes Avr2, Avr4, Avr4E, Avr5 and Avr9 to determine the molecular basis of overcoming Cf genes. Twelve races of C. fulvum with a different virulence spectrum were identified, of which races 9, 2.9, 4.9, 4.5.9 and 4.9.11 occur only in Japan. The Avr genes in many of these races contain unique mutations not observed in races identified elsewhere in the world including (i) frameshift mutations and (ii) transposon insertions in Avr2, (iii) point mutations in Avr4 and Avr4E, and (iv) deletions of Avr4E, Avr5 and Avr9. New races have developed by selection pressure imposed by consecutive introductions of Cf-2, Cf-4, Cf-5 and Cf-9 genes in commercially grown tomato cultivars. Our study shows that molecular variations to adapt to different Cf genes in an isolated C. fulvum population in Japan are novel but overall follow similar patterns as those observed in populations from other parts of the world. Implications for breeding of more durable C. fulvum resistant varieties are discussed
The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions
Karimi Jashni, M. ; Mehrabi, R. ; Collemare, J. ; Mesarich, C.H. ; Wit, P.J.G.M. de - \ 2015
Frontiers in Plant Science 6 (2015). - ISSN 1664-462X - 7 p.
cf-2-dependent disease resistance - extracellular serine-protease - l. enhances resistance - class iv chitinases - phytophthora-infestans - cladosporium-fulvum - proteolytic-enzymes - antifungal activity - gene-expression - tomato
Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs) that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.
Novel introner-like elements in fungi are involved in parallel gains of spliceosomal introns
Collemare, J. ; Beenen, H.G. ; Crous, P.W. ; Wit, P.J.G.M. de; Burgt, A. van der - \ 2015
PLoS ONE 10 (2015)6. - ISSN 1932-6203 - 12 p.
daphnia populations - maximum-likelihood - evolution - gene - positions - conservation - selection - sequence - genomes
Spliceosomal introns are key components of the eukaryotic gene structure. Although they contributed to the emergence of eukaryotes, their origin remains elusive. In fungi, they might originate from the multiplication of invasive introns named Introner-Like Elements (ILEs). However, so far ILEs have been observed in six fungal species only, including Fulvia fulva and Dothistroma septosporum (Dothideomycetes), arguing against ILE insertion as a general mechanism for intron gain. Here, we identified novel ILEs in eight additional fungal species that are phylogenetically related to F. fulva and D. septosporum using PCR amplification with primers derived from previously identified ILEs. The ILE content appeared unique to each species, suggesting independent multiplication events. Interestingly, we identified four genes each containing two gained ILEs. By analysing intron positions in orthologues of these four genes in Ascomycota, we found that three ILEs had inserted within a 15 bp window that contains regular spliceosomal introns in other fungal species. These three positions are not the result of intron sliding because ILEs are newly gained introns. Furthermore, the alternative hypothesis of an inferred ancestral gain followed by independent losses contradicts the observed degeneration of ILEs. These observations clearly indicate three parallel intron gains in four genes that were randomly identified. Our findings suggest that parallel intron gain is a phenomenon that has been highly underestimated in ILE-containing fungi, and likely in the whole fungal kingdom.
Identification and functional characterization of proteases and protease inhibitors involved in virulence of fungal tomato pathogens
Karimi Jashni, M. - \ 2015
Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Jerome Collemare; Rahim Mehrabi. - Wageningen : Wageningen University - ISBN 9789462574571 - 183
passalora fulva - plantenziekteverwekkende schimmels - virulentie - proteïnasen - proteïnaseremmers - plant-microbe interacties - genomica - solanum lycopersicum - tomaten - eiwitexpressieanalyse - passalora fulva - plant pathogenic fungi - virulence - proteinases - proteinase inhibitors - plant-microbe interactions - genomics - solanum lycopersicum - tomatoes - proteomics

Pathogens cause disease on both animal and plant hosts. For successful infection and establishment of disease, pathogens need proper weaponry to protect themselves against host defenses and to promote host colonization to facilitate uptake of nutrients for growth and reproduction. Indeed, plant pathogens secrete various types of effector molecules (proteins and secondary metabolites) to manipulate host responses for their own needs. Secreted proteases and protease inhibitors (PIs) are such effector molecules. Proteases can hydrolyze plant defense proteins and PIs can inhibit plant proteases that are part of the host surveillance system. Despite the importance of proteases and PIs secreted by fungal pathogens, little information about their role in virulence is available. The recent advances in genomics, bioinformatics, transcriptomics and proteomics have facilitated identification and functional analysis of proteases and PIs relevant to plant-fungus interactions.

Chapter 1 is an introduction to the thesis outlining the general concept of plant-microbe interactions. It briefly describes the current knowledge of pathogenicity mechanisms employed by fungal plant pathogens and defense mechanisms employed by their host plants. It further introduces proteases and PIs and their potential role in modifying pathogenesis-related (PR) proteins to facilitate fungal virulence. It completes with an outline of the PhD research project.

In chapter 2, we analyzed and compared the number of putatively secreted proteases present in the genomes of 30 fungi with different lifestyles. The analysis showed that fungi with a saprotrophic and hemibiotrophic lifestyle contain more secreted protease genes than biotrophs. Surprisingly, the number of protease genes present in the genome of Cladosporium fulvum, a biotrophic tomato pathogen, is comparable with that of hemibiotrophs and saprotrophs. We analyzed all C. fulvum protease genes both at the transcriptome and proteome level by means of RNA-Seq/RT-qrtPCR and mass spectrometry analyses, respectively. Results showed that many proteases of C. fulvum are not expressed during growth in planta, likely sustaining the biotrophic growth pattern of this fungus.

In chapter 3, using an alignment-based gene prediction tool, we identified pseudogenes containing disruptive mutations (DMs) that likely lead to the production of nonfunctional proteins, including a group of putatively secreted proteases from C. fulvum. Fewer DMs were observed in other fungi including Dothistroma septosporum, a hemibiotrophic pine needle pathogen and close relative of C. fulvum, and suggested that the difference in pseudogenization of proteases between these two pathogens might in part explain their different lifestyle.

In chapter 4, we analyzed the tomato genome and identified 30 candidate chitinases genes, of which six encoded chitin binding domain (CBD)-containing chitinases. Transcriptome and proteome data were collected after inoculation of tomato with several fungal pathogens and allowed the identification of two CBD-chitinases (SlChi2 and SlChi13) with a putative role in protecting tomato against C. fulvum and F. oxysporum f. sp. lycopersici (F. oxysporum), respectively. Purified CBD-chitinases SlChi1, SlChi2, SlChi4 and SlChi13 were incubated with secreted protein extracts (SPEs) from seven fungal tomato pathogens and we could show that SPEs from F. oxysporum, Verticillium dahliae, and Botrytis cinerea modified SlChi1 and SlChi13. LC-MS/MS analysis revealed that incubation with SPE from F. oxysporum removed the N-terminal 37 and 49 amino acids, comprising part and complete CBD domain from SlChi1 and SlChi13, respectively. Removal of the CBD of SlChi1 and SlChi13 by SPE of F. oxysporum reduced the antifungal activity of the two chitinases. We identified a fungal metalloprotease (FoMep1) and a subtilisin serine protease (FoSep1) that synergistically cleaved both SlChi1 and SlChi13. Transgenic F. oxysporum in which the genes encoding these two proteases were knocked out by homologous recombination lost the ability to cleave the two chitinases and were compromised in virulence on tomato compared to the parental wild type. These results suggest an important role of the two chitinases in defense of tomato against this pathogen.

In chapter 5, we searched for host target(s) of the apoplastic effector Avr9 secreted by C. fulvum during infection of tomato. Based on the structural homology of Avr9 with carboxy peptidase inhibitors, we hypothesized that the host target of Avr9 might be apoplastic proteases. To isolate and identify Avr9 targets in apoplastic fluids, we used synthetic biotinylated Avr9, and performed pull-down and far-western blotting assays with apoplastic fluids from tomato inoculated with a C. fulvum race lacking the Avr9 gene. However, we found no specific Avr9-interacting proteins from pull-down complexes analyzed by mass spectrometry or by far-western blotting. Then, we hypothesized that glycosylation of Avr9 might be required for its biological function. The results of mass spectrometry analysis revealed that Avr9 is N-glycosylated when secreted by C. fulvum, containing at least two GlcNac and six mannose residues. The necrosis-inducing activity of glycosylated and non-glycosylated Avr9 was assayed but appeared not significantly different; however, we could not produce sufficient amounts of (biotinylated)-glycosylated Avr9 to perform pull-down assays for identification of potential glycosylated Arv9-interacting proteins by mass spectrometry.

Previous studies as well as the results present in this PhD thesis showed that fungal pathogens secrete a plethora of effectors including proteases and PIs. Many of identified proteases and PIs mediate effector-triggered immunity in host plants. In chapter 6, we reviewed the recent advances on the various roles of proteases and PIs in compromising basal defense responses induced by microbe-associated molecular patterns.

Chapter 7 is a summarizing discussion of the PhD thesis. We showed determinative roles of proteases and PIs in shaping plant-pathogen interactions. The expression and pseudogenization studies on proteases of C. fulvum showed that the genome content does not necessarily reflect the lifestyle of this fungus. This is true for many classes of fungal genes, including proteases. Fungi contain many different types of proteases whose functions may partly overlap. This hampers the discovery of their biological functions. We could demonstrate that two different types of proteases (metalloprotease (FoMep1) and subtilisin serine protease (FoSep1)) of F. oxysporum act synergistically to modify and reduce antifungal activity of two plant CBD-chitinases. Identifying additional proteases is achievable by a targeted proteomics approach using known targets as we did in chapter 4. However, identification of biological functions of proteases is a technical challenge when targets are not known. Multi-gene targeting of protease and PI genes is required to reveal their function in plant-pathogen interactions, which can only be addressed by using advanced genetic tools in future research.

Synergistic action of serine- and metallo-proteases from Fusarium oxysporum f. sp. lycopersici cleaves chitin-binding tomato chitinases, reduces their antifungal activity and enhances fungal virulence
Karimi Jashni, M. ; Dols, I. ; Iida, Y. ; Boeren, S. ; Beenen, H.G. ; Mehrabi, R. ; Collemare, J. ; Wit, P.J.G.M. de - \ 2015
Molecular Plant-Microbe Interactions 28 (2015)9. - ISSN 0894-0282 - p. 996 - 1008.
As part of their defence strategy against fungal pathogens, plants secrete chitinases that degrade chitin, the major structural component of fungal cell walls. Some fungi are not sensitive to plant chitinases because they secrete chitin-binding effector proteins that protect their cell wall against these enzymes. However, it is not known how fungal pathogens that lack chitin-binding effectors overcome this plant defence barrier. Here, we investigated the ability of fungal tomato pathogens to cleave chitin-binding domain (CBD)-containing chitinases and its effect on fungal virulence. Four tomato CBD-chitinases were produced in Pichia pastoris and incubated with secreted proteins isolated from seven fungal tomato pathogens. Of these, Fusarium oxysporum f. sp. lycopersici, Verticillium dahliae and Botrytis cinerea were able to cleave the extracellular tomato chitinases SlChi1 and SlChi13. Cleavage by F. oxysporum removed the CBD from the N-terminus, as shown by mass spectrometry, and significantly reduced the chitinase and antifungal activity of both chitinases. Both secreted metallo-protease FoMep1 and serine protease FoSep1 were responsible for this cleavage. Double deletion mutants of FoMep1 and FoSep1 of F. oxysporum lacked chitinase cleavage activity on SlChi1 and SlChi13 and showed reduced virulence on tomato. These results demonstrate the importance of plant chitinase cleavage in fungal virulence.
Intron gains through genomic invasion of Introner-Like Elements in fungi
Burgt, A. van der; Wit, P.J.G.M. de; Collemare, J. - \ 2015
In: Book of Abstracts 28th Fungal Genetics Conference. - - p. 152 - 153.
Introner-Like Elements (ILEs) are invasive spiceosomal introns that were identified in six fungal species, where they represent the vast majority of recent intron gains [1]. ILEs differ from Regular Spliceosomal Introns (RSIs) by their longer length and higher stability. Yet, they rapidly degenerate in length and sequence to become undistinguishable from RSIs. It was hypothesized that ILEs are the major mechanism of intron gains in fungi [2]. However, this hypothesis is not supported by their restricted taxonomic distribution. Here, we report the identification of about 10,000 typical ILEs in 53 fungal species that belong to distant classes. Intron gain analyses showed that 96% of inspected ILEs are gained introns, confirming our previous results. Remarkably, we found evidence that new ILEs can originate from other ILEs through sequence insertion, deletion and mutations. Especially in the class of Dothideomycetes, long ILEs originate from conserved short elements. Sequence analysis of these short elements revealed the presence of a motif that might be responsible for their ability to multipy because it is more conserved than splicing signals. However, while splicing signals are constrained, this conserved motif rapidly degenerates to become unidentifiable. This conserved motif could also be identified in most of long ILEs. However, in Sordariomycetes and Leotiomycetes, the motif is slightly different, which may indicate independent origins of ILEs. Altogether, our results show that ILEs are widespread in fungi and regularly emerge to give bursts of intron gains. 1. van der Burgt A, Severing E, de Wit PJ, Collemare J. 2012. Curr Biol. 22(13):1260-5. 2. Collemare J, van der Burgt A, de Wit PJ. 2013. Commun Integr Biol. 6(2):e23147.
Automated alignment-based curation of gene models in filamentous fungi
Burgt, A. van der; Severing, E.I. ; Collemare, J.A.R. ; Wit, P.J.G.M. de - \ 2014
BMC Bioinformatics 15 (2014). - ISSN 1471-2105 - 13 p.
pathogen fusarium-graminearum - ab-initio - genome - prediction - introns
Background Automated gene-calling is still an error-prone process, particularly for the highly plastic genomes of fungal species. Improvement through quality control and manual curation of gene models is a time-consuming process that requires skilled biologists and is only marginally performed. The wealth of available fungal genomes has not yet been exploited by an automated method that applies quality control of gene models in order to obtain more accurate genome annotations. Results We provide a novel method named alignment-based fungal gene prediction (ABFGP) that is particularly suitable for plastic genomes like those of fungi. It can assess gene models on a gene-by-gene basis making use of informant gene loci. Its performance was benchmarked on 6,965 gene models confirmed by full-length unigenes from ten different fungi. 79.4% of all gene models were correctly predicted by ABFGP. It improves the output of ab initio gene prediction software due to a higher sensitivity and precision for all gene model components. Applicability of the method was shown by revisiting the annotations of six different fungi, using gene loci from up to 29 fungal genomes as informants. Between 7,231 and 8,337 genes were assessed by ABFGP and for each genome between 1,724 and 3,505 gene model revisions were proposed. The reliability of the proposed gene models is assessed by an a posteriori introspection procedure of each intron and exon in the multiple gene model alignment. The total number and type of proposed gene model revisions in the six fungal genomes is correlated to the quality of the genome assembly, and to sequencing strategies used in the sequencing centre, highlighting different types of errors in different annotation pipelines. The ABFGP method is particularly successful in discovering sequence errors and/or disruptive mutations causing truncated and erroneous gene models. Conclusions The ABFGP method is an accurate and fully automated quality control method for fungal gene catalogues that can be easily implemented into existing annotation pipelines. With the exponential release of new genomes, the ABFGP method will help decreasing the number of gene models that require additional manual curation.
The role of effectors, pamps, and secondary metabolites in adaptation of Cladosporium fulvum to tomato
Wit, P.J.G.M. de; Mesarich, C.H. ; Ökmen, B. ; Burgt, I.A. van der; Iida, Y. ; Battaglia, E. ; Beenen, H.G. ; Griffiths, S.A. ; Bradshaw, R.E. ; Collemare, J.A.R. - \ 2014
In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 41 - 41.
CS-28.1 - The biotrophic tomato pathogen Cladosporium fulvum is a Dothideomycete fungus that is most related to the hemi-biotrophic fungal pine pathogen Dothistroma septosporum. We are interested in understanding genomic adaptations in these and related fungi that can explain host plant-specificity. The C. fulvum genomic sequence allowed us to identify and study its complete effector catalogue of which many effectors had been identified already and of which three represent core effectors (Avr4, Ecp2 and Ecp6) occurring in several genera and species of fungal plant pathogens. The genome of D. septosporum harbors the highest number of homologs of C. fulvum effectors, but some are pseudogenized. Both C. fulvum and D. septosporum contain a large number of genes involved in the production of secondary metabolites. C. fulvum seems to adapt its host plant tomato by down-regulation or pseudogenization of genes involved in the production of toxic secondary metabolites. One clear example is dothiostromin, a toxin produced by D. septosporum during colonization of pine needle. C. fulvum contains all genes required for the production of this toxin, but a few crucial genes have been pseudogenized. Apart from new effectors (Avr5) we have also identified new pathogen-associated molecular patterns (PAMPs), as well as damage-associated molecular patterns (MAMPs) from C. fulvum. Both represent cell-wall degrading enzymes produced by C. fulvum during infection of tomato. We also found that C. fulvum has adapted to grow on tomato by secreting the enzyme a- tomatinase that hydrolyses the antifungal tomato saponin a-tomatine into the non-toxic compounds lycotetraose and tomatidine.
Secondary metabolism and biotrophic lifestyle in the tomato pathogen Cladosporium fulvum: from comparative genomics to reconstruction of biosynthetic pathways
Collemare, J.A.R. ; Griffiths, S.A. ; Cox, R. ; Wit, P.J.G.M. de - \ 2014
In: Book of Abstracts 10th International Mycological Congress. - - p. 41 - 41.
A fungal biotrophic lifestyle has been associated with a reduction of the potential for production of secondary metabolites (SMs), which are in many cases toxic on the host plants. Analysis of the genome of Cladosporium fulvum, a biotrophic fungal pathogen that causes leaf mould of tomato, challenged this correlation. Indeed, C. fulvum shows a high potential for SM production, including compounds toxic on plants such as elsinochrome, with 23 core genes (10 PKSs, 10 NRPSs, 2 hybrid PKS-NRPSs and 1 DMATS) [1]. However, we showed that only two core genes, PKS6 and NPS9, show high expression in planta, but both are significantly down regulated during colonization of the mesophyll tissue [2]. This result suggests that down-regulation and low expression of SM biosynthetic pathways is another mechanism associated with biotrophy. Functional analysis of PKS6 has now confirmed this hypothesis. Comparative genomics of C. fulvum biosynthetic pathways identified two gene clusters (PKS1 and PKS6) that are conserved across Pezizomycotina. PKS1 belongs to the elsinochrome gene cluster characterized in Elsinoe fawcettii and PKS6 to the endocrocin/monodictyphenone gene cluster characterized in Aspergillus species. We are using several complementary approaches in order to determine SMs produced by these pathways in C. fulvum: i) deletion mutants; ii) over-expression of local transcription factors; iii) heterologous expression in Aspergillus oryzae. We are also determining the evolutionary history of both gene clusters in Pezizomycotina. Altogether, our recent studies suggest that some well-studied biosynthetic pathways might be conserved and ancestral, but the final compounds are diverse with different biological activities. [1] de Wit PJ, van der Burgt A, Ökmen B, Stergiopoulos I, Abd-Elsalam KA, et al., 2012. PLoS Genetics. 8(11):e1003088. [2] Collemare J, Griffiths S, Iida Y, KarimiJashni M, et al., 2014. PLoS One. 9(1):e85877.
Dothideomycete Plant Pathogens Require Specific Virulence Factors for Colonization and Host Plants Have Developed Specific R Genes for Defence
Wit, P.J.G.M. de; Mesarich, C.H. ; Ökmen, B. ; Burgt, A. van der; Iida, Y. ; Battaglia, E. ; Beenen, H.G. ; Griffiths, S.A. ; Collemare, J.A.R. ; Bradshaw, R.E. - \ 2014
In: Book of Abstracts Joint Meeting American Phytopathological Society and Canadian Phytopathological Society. - - p. 44 - S.
The tomato pathogen Cladosporium fulvum is a Dothideomycete that is most related to the hemi-biotrophic fungal pine pathogen Dothistroma septosporum. We study genomic adaptations in these and related fungi that can explain host plant-specificity. The C. fulvum genomic sequence allowed us to identify and study its complete effector catalogue including three core effectors (Avr4, Ecp2 and Ecp6). The genome of D. septosporum harbors the highest number of homologs of C. fulvum effectors, but some are pseudogenized. Both C. fulvum and D. septosporum contain a large number of genes involved in the production of secondary metabolites. C. fulvum seems to adapt its host plant tomato by down-regulation or pseudogenization of genes involved in the production of toxic secondary metabolites. One example is dothiostromin, a toxin produced by D. septosporum during colonization of pine needle. C. fulvum contains all genes required for the production of this toxin, but a few of these genes are pseudogenized. Apart from new effectors (Avr5) we have also identified new pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) from C. fulvum. Both represent cell-wall degrading enzymes produced by C. fulvum during infection of tomato. We also found that C. fulvum has adapted to grow on tomato by secreting the enzyme a-tomatinase that hydrolyses the antifungal tomato saponin a-tomatine into the non-toxic compounds lycotetraose and tomatidine.
Role of effector proteins secreted by Cladosporium fulvum againist the mycoparasitic invasion
Iida, Y. ; Ökmen, B. ; Karimi Jashni, M. ; Mesarich, C.H. ; Ikeda, K. ; Collemare, J.A.R. ; Wit, P.J.G.M. de - \ 2014
In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 81 - 81.
P347 - Most fungal pathogens secrete effector molecules that function as virulence factors to facilitate disease on hosts, but they are also recognized by cognate plant resistance proteins to arrest fungal growth. Ten effector genes identified in leaf mold pathogen Cladosporium fulvum are strongly up-regulated during infection of the host plant tomato, but not or hardly in vitro. The fungal mycoparasite Dicyma puvinata is a well-known biocontrol agent that parasitizes several fungal leaf pathogens including C. fulvum. We found that in C. fulvum expression of most effector genes is up-regulated when it is co-cultured with D. puvinata. In addition, C. fulvum ¿ Avr4 and ¿Ecp6 deletion mutants are more susceptible to D. pulvinata than wild-type strain, suggesting that these effectors are not only important for defence of C. fulvum against tomato chitinases during infection but also to defend this fungus against mycoparasitic invasion. Most effectors are species-specific and a few are non-species-specific core effectors like Avr4 and Ecp6. Avr4 protects the C. fulvum cell wall from hydrolysis by tomato chitinase directly through chitin binding, whereas Ecp6 scavenges chitin fragments in the apoplastic region to prevent chitin-mediated elicitation of immune responses. These results suggest that chitinases from D. pulvinata are important for mycoparasitism, and C.fulvum deploys the same effectors to protect itself against both plants and mycoparasites.
Identification of fungal proteases responsible for proteolytic cleavage of tomato chitinases
Karimi Jashni, M. ; Mehrabi, R. ; Collemare, J.A.R. ; Wit, P.J.G.M. de - \ 2014
In: Book of Abstracts XVI International Congress on Molecular Plant-Microbe Interactions. - - p. 21 - 21.
CS-5.4 - Plants defend themselves against fungal pathogens by secreting enzymes with antifungal activities such as chitinases that degrade the fungal cell wall. In response, fungal pathogens secrete chitin-binding proteins such as Avr4 which can protect them against plant chitinases. This protection might be incomplete, so released chitin oligomers may still be detected by plant recognition receptors and induce defence. Fungi like C. fulvum secrete Ecp6 which can sequester chitin oligomers preventing them to be detected. Previously, degradation of PR proteins has been proposed as a component of virulence of some plant pathogens. Here, we employed a combined biochemical, proteomics, bioinformatics and mass spectrometry approach to identify the chitinase modifying protein(s). We have produced four chitin binding tomato chitinases (CBD-tomato chitinases) in Pichia pastoris and analysed eight tomato fungal pathogens for their ability to degrade their CBD-. We showed that enzymes present in culture filtrate of some of these tomato pathogens can degrade two of the four CBD-tomato chitinases. Culture filtrates with proteolytic activity were fractionated on Superdex 75 and analysed. Mass spectrometry confirmed the presence of at least two novel secreted serine proteases in the active fractions. We also performed expression profiling for the fungal proteases and CBD-tomato chitinases during infection of susceptible and resistant tomato cultivars. In addition, we are testing whether the two serine proteases play a role in virulence.
Functional analysis of the conserved transcriptional regulator CfWor1 in Cladosporium fulvum reveals diverse roles in the virulence of plant pathogenic fungi
Ökmen, B. ; Collemare, J. ; Griffiths, S.A. ; Burgt, A. van der; Cox, R. ; Wit, P.J.G.M. de - \ 2014
Molecular Microbiology 92 (2014)1. - ISSN 0950-382X - p. 10 - 27.
avirulence gene avr9 - dna-binding domains - candida-albicans - alternaria-brassicicola - magnaporthe-grisea - master regulator - expression - tomato - family - penetration
Fungal Wor1-like proteins are conserved transcriptional regulators that are reported to regulate the virulence of several plant pathogenic fungi by affecting the expression of virulence genes. Here, we report the functional analysis of CfWor1, the homologue of Wor1 in Cladosporium fulvum. ¿cfwor1 mutants produce sclerotium-like structures and rough hyphae, which are covered with a black extracellular matrix. These mutants do not sporulate and are no longer virulent on tomato. A CE.CfWor1 transformant that constitutively expresses CfWor1 produces fewer spores with altered morphology and is also reduced in virulence. RNA-seq and RT-qrtPCR analyses suggest that reduced virulence of ¿cfwor1 mutants is due to global downregulation of transcription, translation and mitochondrial respiratory chain. The reduced virulence of the CE.CfWor1 transformant is likely due to downregulation of effector genes. Complementation of a non-virulent ¿fosge1 (Wor1-homologue) mutant of Fusarium oxysporum f. sp. lycopersici with CfWor1 restored expression of the SIX effector genes in this fungus, but not its virulence. Chimeric proteins of CfWor1/FoSge1 also only partially restored defects of the ¿fosge1 mutant, suggesting that these transcriptional regulators have functionally diverged. Altogether, our results suggest that CfWor1 primarily regulates development of C.¿fulvum, which indirectly affects the expression of a subset of virulence genes.
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